Peptides derived from foreign and self antigens are presented in the context of MHC class II molecules for recognition by CD4+ T cells. Class II molecules transit the endosomal network of cells, acquiring peptides from exogenous antigens as well as endogenous cell surface and endosomal proteins. Surprisingly, epitopes from cytoplasmic antigens also are presented by class II molecules. The overall goal of this proposal is to understand the mechanisms regulating class II cytoplasmic Ag presentation, and to identify new means to modulate immunity to self and tumor Ags. With subversion of class I presentation by tumors and viruses, exploiting novel pathways for cytoplasmic Ag presentation remains important. Targeting specific cytoplasmic Ags for class II presentation offers a unique approach to promote immune recognition and overcome such immune evasion. The current proposal focuses on a selective pathway, chaperone mediated autophagy (CMA) which facilitates class II cytoplasmic antigen presentation. We propose that cytoplasmic antigens are selectively targeted by cytosolic chaperones for proteolysis during CMA. The resulting peptides may then be chaperoned to a membrane transporter, LAMP-2A for translocation into the endosomal network for class II presentation. Studies are proposed to define the importance of chaperones, HSC70 and HSP90 in guiding cytoplasmic antigens for processing and translocation into organelles for class II presentation. Multiple isoforms of the LAMP-2 gene are expressed in B cells and melanomas, potentially regulating class II cytoplasmic antigen presentation by autophagy. Studies are proposed to elucidate the role of individual LAMP- 2 isoforms in class II presentation pathways. This project will offer critical insights into the molecular events and essential co-factors for CMA which faciliate class II access to cytoplasmic antigens for immune recognition. Notably, autophagy pathways are often up-regulated in tumor cells to promote tumor growth and survivial. Such changes in cellular autophagy pathways may enhance tumor antigen recognition. Studies in this project will examine the importance of distinct autophagy pathways in cytoplasmic antigen presentation by melanomas. This work also has relevance to our understanding of autoimmunity to self antigens and tolerance induction. Exploiting the selectivity of the CMA pathway may also offer opportunities for novel approaches to vaccine development.